Rho GTPases belong to the family of the 20 small GTPases homologous to Ras which are largely considered as undrugable proteins. Their physiologic activity resides more in a conformational change of the switch I and switch II highly conserved domains than in their guanine nucleotide triphosphate hydrolase very slow catalytic activity. Small G proteins are therefore molecular switch that cycle between an inactive GDP bound state an active GTP bound conformation. The Rho subfamily, which contains RhoA, RhoB and RhoC that share more than 85% sequence identities are pleiotropic proteins involved in a wide range of major cell processes, including the control of actomyosin cytoskeleton, cell adhesion, cytokinesis, cell migration, stress response as well as cell survival or apoptosis. These GTPases are post-translationally modified by addition of a carboxy terminal isoprenoid group necessary for their anchorage to cellular membranes, where they can be activated upon various stimuli by an exchange of nucleotide catalyzed by Guanine nucleotide Exchange Factors (GEF). Activation step is then counterbalanced by GTP hydrolyses which is enhanced by GTPase Activating Proteins (GAPs). Therefore the cellular pool of activated Rho is maintained at the cellular membrane to limited pool. The major fraction is extracted from the membrane and sequestered in the cytoplasm by Guanine nucleotide Dissociation Inhibitors (GDI). These large proteins interact by their amino terminal part with the switch domains of Rho, thus preventing the release of the GDP, and also by their carboxy terminal part to the isoprenyl group, shielding this hydrophobic moiety in order to maintain the Rho proteins soluble while excluded from membranes.
Together these regulators maintain the largest fraction of Rho Proteins inactive in the cell, often as much as 95%, in order to quickly activate a very small population that will interact with effector proteins to initiate cellular transduction pathways. Moreover study of the crosstalk between the 3 GDI and all interacting Rho revealed that overexpression of a single Rho can induce artificially displacement and degradation of others and impair signaling pathways not directly controlled by the transgene. This critical point highlights the complexity of targeting individual Rho in a selective manner.
The peculiar RhoB seems to be involved in different cellular functions and regulations than its closest homologs RhoA and RhoC. RhoB can be palmitoylated and either farnesylated or geranylgeranylated, prenylation which define localization to the plasma membrane or the endosome respectively. Some main RhoB functions in intracellular trafficking and adhesion have been characterized using conventional molecular tools such as overexpression of wild type or mutants or genetic knock down by RNA interference. Other functions have been connected to RhoB gene expression as an immediate early response to cytokines or growth factors as well as DNA damaging agent or radiation. In addition, RhoB plays paradoxal roles in cancer progression. RhoB can alter tumor formation and is often down regulated in head and neck or lung cancers.
Nevertheless RhoB is also promoting tumor angiogenesis and protecting from apoptosis in cells with genomic instability. There are now clear evidences that RhoB exert pleiotropic functions which are cells and context dependent. Previous studies targeted RhoB at the genetic level by overexpression, RNAi or gene knock out in mice. However these methods altered RhoB functions in a global way, knocking down all RhoB activities in cells, but mostly altering both the GDP bound major fraction which can induce imbalances in the GDI-Rho interactions and the minor GTP bound active pool. To decipher RhoB function without interfering with other Rho activities, it would be necessary to target RhoB at the protein level. Albeit there are no small molecule inhibitors targeting Rho GTPases, the C3 exoenzyme from Claustridium botulinum or Bacillus cereus are natural inhibitors that induce ADP-ribosylation of Rho, preventing their activation by GEF and further increasing the binding of free Rho to GDI. Actually expression of C3 gene in eukaryotic cells or incubation with cell permeable tat-C3 has been successfully used to alter globally the function of all 3 Rho, leading to a strong phenotype of actin fiber loss and cell rounding. Several other bacterial toxin target as well Rho proteins. Nevertheless all these toxins lack specificity because they do not discriminate between RhoA, RhoB or RhoC and mostly do not block directly the activated form of Rho.
In some previous studies, the selection of recombinant single chain antibodies from phage display libraries was established in order to identify binding molecules selective to the active GTP bound state of Rho proteins. Actually recombinant antibody selected from large display libraries have been used in many biotechnological or biomedical applications. Although most of recombinant antibodies require for stability the canonical disulfide bond within the VH or VL variable domains, some peculiar intracellular antibodies, referred as intrabodies, remains stable in the reducing environment. Thus, depending on the antibody format, the scaffold properties and the library diversities, some rare recombinant antibodies have been reported to be functional while expressed in the cytosol of eukaryotic cells (Tanaka, T., Williams, R. L. & Rabbitts, T. H. Tumour prevention by a single antibody domain targeting the interaction of signal transduction proteins with RAS. EMBO J 26, 3250-3259, doi:7601744 [pii] 10.1038/sj.emboj.7601744 (2007); Nizak, C. et al. Recombinant antibodies to the small GTPase Rab6 as conformation sensors. Science 300, 984-987, doi:10.1126/science.1083911 (2003); Meli, G., Visintin, M., Cannistraci, I. & Cattaneo, A. Direct in vivo intracellular selection of conformation-sensitive antibody domains targeting Alzheimer's amyloid-beta oligomers. J Mol Biol 387, 584-606, doi:10.1016/j.jmb.2009.01.061 (2009)). The first active Rho conformational single chain variable fragment (scFv), named scFvC1, recognized in vitro in biochemical assays the GTP bound state of all 3 Rho (Goffinet, M. et al. Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection. BMC Biotechnol 8, 34, doi:1472-6750-8-34 [pii] 10.1186/1472-6750-8-34 (2008).). A molecular evolution of scFvC1 led to the identification of the scFvF7, a higher affinity pan active Rho binder than scFvC1, as well as the scFvE3 that preferentially recognizes active RhoB but is not functional as an intrabody.